Abstract

N-Acetyltransferase 1 (NAT1), encoded by the polymorphic NAT1 gene, has been shown to be one of the major enzymes in human breast tissue that activates aromatic and heterocyclic amines. Humans are mainly exposed to these carcinogens through cigarette smoking and consumption of well-done meat. To test the hypothesis that variations in the NAT1 gene are related to breast cancer risk, particularly among women who smoke or consume high levels of well-done meat, a nested case-control study was conducted in a prospective cohort study of 41,837 postmenopausal Iowa women. Information on cigarette smoking and other breast cancer risk factors was obtained at the baseline survey conducted in 1986. DNA samples and information on the consumption of well-done meat were obtained, in the case-control study, from breast cancer cases diagnosed from 1992 to 1994 and a random sample of cancer-free cohort members. Genomic DNA samples obtained from 154 cases and 330 controls were assayed for 11 NAT1 alleles (NAT1*3, *4, *5, *10, *11, *14, *15, *16, *17, *19, and *22). The NAT1*4 allele was the predominant allele observed in this study population, accounting for 73.2% (72.4% in cases versus 73.8% in controls) of the total alleles analyzed. Compared to controls, breast cancer cases had a slightly higher frequency of the NAT1*10 allele (18.8% in cases versus 17.3% in controls) and a substantially higher frequency of the NAT1*11 allele (3.6% versus 1.2%). In multivariate analyses, we found a 30% [95% confidence interval (CI) = 0.8–1.9] elevated risk of breast cancer associated with the NAT1*10 allele and a nearly 4-fold (95% CI = 1.5–10.5) elevated risk associated with the NAT1*11 allele. The positive association of breast cancer with the NAT1*11 allele was more evident among smokers [odds ratio (OR) = 13.2, 95% CI = 1.5–116.0] and those who consumed a high level of red meat (OR = 6.1, 95% CI = 1.1–33.2) or consistently consumed their red meat well done (OR = 5.6, 95% CI = 0.5–62.7). The association of the NAT1*10 allele with breast cancer was mainly confined to former smokers (OR = 3.3, 95% CI = 1.2–9.5). These findings are consistent with a role for the NAT1 gene in the etiology of human breast cancer.

Introduction

Aromatic and heterocyclic amines have been shown in animal studies to induce tumors of various organs (1, 2, 3)
, including those of the mammary glands (2, 3, 4)
. Humans are mainly exposed to these carcinogens through cigarette smoking and consumption of well-done meat (5, 6, 7, 8)
. We have shown recently in a case-control study that well-done meat intake may be an important risk factor for breast cancer (9)
. Similar findings have been reported elsewhere, although there are some inconsistencies among the studies (10, 11, 12, 13, 14)
. The results on the association between cigarette smoking and breast cancer risk have been conflicting, with some studies reporting increased risk of breast cancer among smokers and others reporting no association or even a reduced risk (15, 16, 17, 18, 19)
.

Part of the inconsistencies observed in previous studies may reflect that only a few of them have investigated potential modifying effects of relevant carcinogen-metabolizing enzymes on the associations of breast cancer with cigarette smoking and/or consumption of well-done meat (20, 21, 22, 23)
. N-Acetyltransferases are believed to be important in the activation and detoxification of aromatic and heterocyclic amines (24, 25, 26)
. In humans, two N-acetyltransferases (NAT15
and NAT2) have been identified ,(26, 27, 28)
. The NAT2 gene is known to be polymorphic in humans (26, 27)
. Recently, the NAT1 gene has also been shown to be polymorphic (29)
. Both NAT1 and NAT2 have been shown to catalyze the metabolic activation of aromatic and heterocyclic amine carcinogens (24, 30, 31, 32)
. Several epidemiological studies, including our own study among postmenopausal Iowa women (23)
, have evaluated the association between NAT2 polymorphisms and breast cancer risk, but the results from these studies have been inconclusive (20, 21, 22, 23, 33)
. In 1996, Sadrieh et al.(34)
reported that NAT1 but not NAT2 activity was detectable in human mammary epithelial cells. They further showed that human mammary gland catalyzed the metabolic activation of heterocyclic amines, suggesting that NAT1 may be more important than NAT2 in the etiology of breast cancer. Prompted by this observation and findings from recent studies showing the potential etiological importance of NAT1 in other human cancers (35, 36)
, we conducted a case-control study among participants in the IWHS to investigate whether NAT1 polymorphisms associate with breast cancer risk and, further, whether the association is modified by cigarette smoking or consumption of well-done meat.

Materials and Methods

IWHS.

Detailed descriptions of this cohort study have been published elsewhere (37, 38, 39)
. Briefly, 41,836 Iowa women, ages 55–69 years, who completed a mailed questionnaire in January 1986, have been followed for mortality and cancer incidence. Virtually all IWHS participants were Caucasian women. The follow-up was accomplished through computer linkage of study participants with Iowa death certificate files, the National Death Index, and cancer diagnosis data collected by the Iowa State Health Registry, part of the Surveillance, Epidemiology, and End Results Program of the National Cancer Institute. The self-administered questionnaire used in the 1986 baseline survey included information on diet and other major risk factors for cancer, such as cigarette smoking, reproductive factors, family history of cancer, prior medical conditions, and hormone use. At baseline, however, no blood sample was collected, and no information was obtained on usual intake of meat, cooking method, or usual doneness levels of meat. A case-control study with a supplementary survey to obtain information on consumption of meat and usual doneness level (see below) and DNA sample collection was conducted during 1995–1996 in a subset of cohort members.

Case-Control Study of Breast Cancer.

Eligible cases for this study included all IWHS cohort members who had breast cancer diagnosed from January 1, 1992, to December 31, 1994 (n = 453). A control sample of 900 women was randomly selected from cohort members who were alive and free of cancer on January 1, 1992. Of these 900 women, 24 were excluded from the control group because they were either later found to have a breast cancer diagnosis during the 1992–1994 period (n = 3) or were selected to participate in other IWHS ancillary projects (n = 21). The design of the study was approved by the Institutional Review Board Human Subjects Committee at the University of Minnesota and the Iowa State Health Registry. Informed consent was obtained from all women who participated in this study.

All eligible subjects were asked to complete a self-administered semiquantitative food frequency questionnaire on meat consumption during the “reference” year. Three reference years were identified (1991, 1992, and 1993), corresponding, for cases, to the years immediately prior to breast cancer diagnosis. Women in the control group of the study were also divided randomly into three groups with approximately equal sample sizes to assess their dietary habits during the same three reference years. The food questionnaire included questions on the usual consumption and preparation methods of various meats. In addition, participants reported their usual preference for level of meat doneness using a series of color photographs that represented increasing levels of doneness of hamburger (four photos), beefsteak (four photos), and bacon (three photos). Photographs were labeled by number only and represented a range of doneness levels from extremely rare to very well done. Two hundred seventy-three (60.3%) of the selected cases and 659 (75.0%) controls participated. The major reasons for nonparticipation were refusal (29.1% of cases and 18.7% of controls), inability to locate (4.9% of cases and 3.8% of controls), or death prior to contact (5.7% of cases and 2.5% of controls).

Blood samples were donated by a subset of the women who participated in this supplementary mail survey. A blood collection kit, consisting of vacutainer tubes, biological specimen packaging containers and envelopes, and instructions, was mailed to all women who agreed to donate a blood sample. Study participants were instructed to contact their physicians, have their blood drawn, and return samples via express mail using preaddressed, prepaid envelopes provided by the study. Of the 930 women who completed the survey questionnaire, 488 (156 cases and 332 controls) donated a blood sample to the study, giving a response rate of 52.5% (57.1% for cases and 49.9% for controls). Blood was drawn using vacutainers containing acid citrate dextrose solution (Becton Dickinson, Franklin Lakes, NJ). Genomic DNA was extracted from peripheral blood leukocytes according to standard methods and stored at −70°C for subsequent assays.

NAT1 genotypes were determined by a PCR RFLP-based assay (40)
that differentiates among eight NAT1 alleles (NAT1*3, *4, *5, *10, *11, *14, *15, and *16) that were identified previously in human populations. Recently, three new human NAT1 alleles that encode proteins with reduced N-acetyltransferases activity have been identified: NAT1*17, *19, and *22(41, 42)
. NAT1*17 contains a C190→T substitution, NAT1*19 contains a C97→T substitution, and NAT1*22 contains an A752→T substitution. To distinguish among these new NAT1 alleles, the published NAT1 genotyping assay was modified as described below. NAT1 genotype assignments were blind to case-control status.

Gel electrophoresis following RFLP analysis of the 1158-bp NAT1 PCR product. Lane M, molecular weight marker φX174/HaeIII. The restriction digest with BanI and Bg/II indicates that the sample in Lane 4 is heterozygous for the A752→T substitution. The remaining samples do not have the A752→T substitution on either allele. Additionally, no samples in Lanes 1–8 have the C97→T or G350,351→C substitution on either allele.

Gel electrophoresis following RFLP analysis of the C190→T nested PCR product. Lane M , molecular weight marker φX174/HaeIII; Lane C, sample homozygous for the C190→T substitution used here as a positive control. EcoRI digestion results indicate that the sample in Lane 3 is heterozygous for the C190→T substitution. The remaining samples do not have the C190→T substitution on either allele.

Of the 488 DNA samples included in the study, 4 did not amplify and 2 contained alleles that were different from those under study. Therefore, the data analysis was based on 154 cases and 328 controls. ORs were used to measure the strength of the association between exposures and cancer risk. Unconditional logistic regression was used to control for potential confounders assessed at the baseline survey and to derive adjusted ORs and 95% CIs. Red meat intake and doneness levels of these meats were used to estimate exposure to well-done meat. Intakes (in g) of hamburgers, cheeseburgers, beefsteaks, pork chops, bacon, breakfast sausage links, breakfast sausage patties, other sausages, bratwurst, and hot dogs or franks were summed to form the red meat variable. Women who reported eating hamburger, beefsteak, and bacon consistently at well- or very well-done levels were classified as the “exposed” group. These variables, along with variables on cigarette smoking, were analyzed in combination with NAT1 genotype to evaluate potential gene-environment interactions in the etiology of breast cancer.

Results

Table 1<$REFLINK>
shows the case-control distribution and ORs for selected demographic variables and major risk factors identified for this cohort of women in the early years of follow-up. Information related to cigarette smoking and well-done meat intake, the main effects studied here, is also presented in Table 1<$REFLINK>
. Among women who completed the supplementary survey, the risk of breast cancer was found to be positively associated with aging, education, family history of breast cancer, estrogen use, waist:hip ratios, smoking, and well-done meat intake, although not all elevated ORs were statistically significant. With only a few exceptions, similar associations were observed among women who provided a blood sample to the study.

Comparison of cases and controls by selected demographic and risk factors among postmenopausal Iowa women

The allele frequencies of the NAT1 gene are presented in Table 2<$REFLINK>
. There was no statistically significant difference in the overall distribution of allele frequency between cases and controls. NAT1*4, the wild-type allele, presented in nearly three-quarters of the chromosomes tested, and its frequency was comparable between cases and controls. Slightly more cases (18.8%) than controls (17.3%) had the NAT1*10 allele. Other NAT1 alleles were relatively uncommon in this study population. Of note, the NAT1*11 frequency was three times more common in cases (3.6%) than in controls (1.2%).

Distribution of NAT1 alleles by cases and controls among postmenopausal Iowa women

The frequency distribution of specific NAT1 genotypes by case-control status is presented in Table 3<$REFLINK>
. Because of a small sample size and relatively low frequency of most NAT1 variants, ORs were calculated for selected genotype groups (Table 4)<$REFLINK>
. The NAT1*3 allele was uncommon, and this allele associated primarily with the wild-type NAT1*4 to form the heterozygous NAT1*3/*4 genotype. Therefore, NAT1*3/*3, NAT1*3/*4, and NAT1*4/*4 were combined into one group and used as the reference group in the OR estimates of other genotypes. A 30% increase in breast cancer risk was associated with the NAT1*10/any genotype, but the OR was not statistically significant. No elevated risk (OR = 0.8, 95% CI = 0.3–2.4) was found for NAT1*10 homozygotes (5 cases and 13 controls). Eleven cases (7.1%) and 7 controls (2.1%) were NAT1*11 homozygotes or heterozygotes, with a nearly 4-fold significantly elevated risk of breast cancer. Only one NAT1*11 homozygote and two NAT1*10/*11 heterozygotes were identified in this study. Breast cancer was not associated with the presence of NAT1*14, NAT1*15, NAT1*17, or NAT1*22, all of which were rare alleles in this study population. Adjustment for a family history of breast cancer did not change the ORs presented in Table 4<$REFLINK>
, suggesting that these NAT1 alleles may not contribute substantially to the familial clustering of breast cancer among postmenopausal Iowa women.

Adjusted ORs for the associations of breast cancer with NAT1 genotype among postmenopausal Iowa women

To evaluate potential modifying effects of NAT1 polymorphism on the associations of breast cancer with cigarette smoking or consumption of well-done meat, adjusted ORs were calculated according to the joint distribution of selected NAT1 genotypes and exposure levels of these two lifestyle factors (Table 5)<$REFLINK>
. Cigarette smoking was not related to breast cancer risk among the combined NAT1*3/*3, *3/*4, and *4/*4 genotype group. A 13-fold elevated risk (95% CI = 1.5–116.0), however, was observed for cigarette smoking among women with the NAT1*11/any genotype. Among the NAT1*10/any stratum, smoking was associated with a slightly elevated risk (OR = 1.4, 95% CI = 0.7–2.9) of breast cancer when subjects with the NAT1*10/*11 genotype (two cases, no controls) were excluded. When these subjects were included in the analysis, a 60% elevated risk (95%CI = 0.8–3.3) was associated with cigarette smoking. Similarly, inclusion of subjects with the NAT1*10/*11 genotype in the NAT1*10/any stratum increased the OR from 2.2 (95% CI = 0.7–6.6) to 3.3 (95% CI = 1.2–9.5) among those who had quit smoking >10 years ago.

High consumption of red meat was positively associated with the risk of breast cancer only among the NAT1*11/any stratum, with an OR of 6.1 (95% CI = 1.1–33.2) observed in the highest meat intake group. Women who reported consuming red meat at a consistently well-done level were at an increased risk of breast cancer across all genotype groups. The risk was particularly high (OR = 5.6, 95% CI = 0.5–62.7) for women with the NAT1 *11/any genotype who consumed red meat at a consistently well-done level. This OR, however, was not statistically significant, perhaps due to a small sample size.

Discussion

We found in this case-control study that the NAT1*11 allele was associated with an ≈4-fold elevated risk of breast cancer. The risk was increased particularly among women who smoked cigarettes, consumed a high level of red meat, or had a preference for consistently well-done meat. The NAT1*10 allele was related to a slightly elevated risk of breast cancer, and this association was primarily confined to former or light smokers. These findings suggest that the NAT1 gene may play a role in the etiology of breast cancer.

Although the association between the NAT1*11 allele and breast or other human cancers has not been reported previously, the NAT1*10 allele has been linked to increased risk of bladder, colon, gastric, lung, and head and neck cancers in previous case-control studies (35, 36, 43, 44, 45)
. Recently, Millikan et al.(33)
reported no overall association of NAT1 polymorphism with breast cancer risk among African-American and white women. The risk, however, was substantially elevated among postmenopausal women with the NAT1*10 allele who had quit smoking within the past 10 years. Intriguingly, no modifying effect of the NAT1*10 allele was observed among current smokers. That study differed from ours because their NAT1 genotype method distinguishes only four alleles (NAT1*4, NAT1*3, NAT1*10, and NAT1*11) and the NAT1*11 allele was identified in only 1% of cases and 2% of controls among white women and was absent in African-American women.

In humans, two N-acetyltransferases (NAT1 and NAT2) catalyze N- and O-acetylation of various procarcinogens, notably, aromatic and heterocyclic amines (24, 30, 31, 32)
. It is believed that these procarcinogens are activated via O-acetylation and detoxified by N-acetylation (26)
. The specificity of NAT1 and NAT2 in these two acetylation pathways, however, remains unclear. NAT2 is expressed primarily in the liver (46)
. If this enzyme is involved in O-acetylation of aromatic and heterocyclic amines, it may be less likely that the reactive form of these compounds could reach a high level in the breast or other target organs following activation in the liver. Given the extrahepatic distribution of the NAT1 enzyme (34, 47, 48, 49, 50)
, this enzyme may be more important than the NAT2 enzyme in the activation of aromatic and heterocyclic amines in the target organs. Indeed, it has been shown that the O-acetylation of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine and 2-amino-3-methylimidazo-(4,5-f)quinoline, two of the most abundant heterocyclic amines in well-done meat (5, 6)
, is largely carried out by NAT1 in human mammary glands (34)
.

Although the link between NAT1 alleles and isozyme activity has not been directly established (46)
, two studies have suggested that the NAT1*10 allele may be associated with increased NAT1 activity in the bladder and colon (47, 48)
. However, in more recent studies, individuals with the NAT1*10 allele did not reflect a more rapid phenotype in vivo(51)
or in vitro in the colon (52)
. The association of breast cancer with the NAT1*10 allele was weak in our study (OR = 1.3, 95% CI = 0.8–1.9), and the elevated risk was mainly confined to former (OR = 3.3, 95% CI = 1.2–9.5) or light (OR = 5.0, 95% CI = 0.4–56.2) smokers. This observation is consistent with the results from the recent study by Millikan et al.(33)
. The link between the NAT1*11 allele and enzyme expression and activity remains unclear. One study reported that recombinant NAT1 11 catalyzed N-acetylation at rates ≈2-fold higher than NAT1 4 (53)
, whereas a more recent study reported that recombinant NAT1 11 catalyzed N-acetylation at rates comparable to NAT1 4 (51)
. Recently, the coding region of the NAT1*11 allele was sequenced and found to contain a G445→A substitution that had not been described previously (54)
. The G445→A substitution in the NAT1*11 allele causes an amino acid change from valine to isoleucine. Recent studies show that this substitution resulted in a recombinant NAT1 protein that catalyzed the metabolic activation of N-hydroxy aromatic amines by O- and N,O-acetylation at rates up to 2-fold higher than wild-type recombinant human NAT1 (54)
. The results of this study showing elevated risk of breast cancer with the NAT1*11 allele, particularly among women with high exposure to aromatic and heterocyclic amines, are consistent with increased activation of aromatic and heterocyclic amines in individuals carrying the NAT1*11 allele.

The primary concern of this study may be the low response rate to blood sample collection. There is no reason, however, to speculate that the response rate would be associated with both case-control status and certain genotypes. Nevertheless, we have evaluated the possibility of this selection bias by comparing women who provided blood with those who completed the supplementary questionnaire regarding well-done meat intake and found that these two groups were generally comparable in breast risk factors, including cigarette smoking and well-done meat intake. We have also shown previously that women who completed the supplementary survey were similar to all eligible women who where selected for this ancillary study in virtually all major breast cancer risk factors (9)
. Therefore, this potential bias seems minimal. Passive smoking has been linked recently to an elevated risk of breast cancer in some studies (55)
. Information related to passive smoking, however, was not collected in this study.

In summary, this case-control study found that postmenopausal women with the NAT1*11 allele may be at increased risk of breast cancer, particularly if they smoked cigarettes or consumed a high level of well-done meat. The NAT1*10 allele was also associated with an elevated risk among former or light smokers. These findings are consistent with the observations from recent laboratory studies, indicating the importance of the NAT1 enzyme in the in situ activation of heterocyclic amines in human breast epithelial cells, and point to the need in future studies to investigate potential interactions of genetic factors with intake of foods, food constituents, and nutrients in the etiology of cancer (56)
. Our findings, particularly those related to potential gene-environment interaction, are preliminary, however, given the small sample size and relatively low frequency of the NAT1*11 allele in the study population. Therefore, in addition to laboratory studies to link the NAT1*11 allele with its enzyme function, future epidemiological studies are needed to investigate the role of the NAT1 genes in the etiology of breast cancer.

Acknowledgments

We thank Drs. Lawrence Kushi, Kristin Anderson, and Myron Gross for their contributions in data and specimen collection; Dr. Rashmi Sinha for providing copies of the food photographs for assessing meat doneness level. The food frequency questionnaire used in this study was modified from the one developed by Drs. Sinha and Anderson for a case-control study of pancreatic cancer in Minnesota.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by National Cancer Institute Grants R01 CA-39742 and R01 CA-34627 and National Action Plan on Breast Cancer, Office on Women’s Health, United States Public Health Service Supplementary Grant OWH-284.